The epitope tagging is the recombinant DNA method for making the gene product immunoreactive to an already existing antibody (Jarvik and Telmer, Annu. Rev. Genet. 32:601-618, 1998). Typically the process involves inserting the nucleotide sequence encoding the peptide tag into the gene of interest and expressing the gene in an appropriate host. The protein can then be detected and/or purified by virtue of its interaction with the antibody specific to the epitope tag. This approach can elucidate the size of the tagged protein as well as its abundance, cellular location, posttranslational modifications and interactions with other proteins. In particular, antibodies recognizing the peptide tag facilitate purification and/or isolation of tagged proteins.
The epitope tagging is widely used for detecting, characterizing and purifying proteins. The technique offers several advantages over alternative methods of detecting and purifying proteins. The epitope tagging saves the time and resources comparing with the traditional method of producing an antibody to the specific protein being studied. The small size of the epitope tag, which is usually 5-15 amino acids in length, generally has no effect on the biological function of the tagged protein. However, sometimes the epitope tag may interfere with the protein structure and function or the target protein sequence can influence the antigenicity of the epitope. For this reason, it is sometimes essential to develop epitope tags of different sequence characteristics (different net charges, hydrophobicity and side groups) to increase the chance of success in tagging applications.
To date there are number of the epitope tags commercially available. Often they are incorporated into the expression vectors for mammalian, insect, yeast or bacterial cells. A variety of epitope tags are available, including c-myc, FLAG, HA, His6, T7-Tag, HSV-Tag, Pk-Tag, VSV-Tag, Glu-Glu, BTag and S-Tag. Most epitopes that have been popular for the epitope tagging are highly charged (HA, c-myc, FLAG). Since one generally aims to place the tag in the external portion of the target protein, it is appropriate that the tag be charged rather that hydrophobic. However, the tag of extreme or inappropriate charge could cause the problems in some cases, for example, if a basic domain of a protein is tagged with an acidic sequence. The epitope tags without highly charged amino acids are T7-Tag and BTag. The present invention seeks to provide an alternative to the currently available epitope tags. The E2Tag consists of ten 10 amino acid residues, SSTSSDFRDR (SEQ ID No. 1). We have designated this sequence as E2Tag. All these ten amino acids are required and are sufficient for strong interaction with monoclonal antibody 3F12. The first half of the sequence consists of polar amino acids, and the second half contains charged amino acid residues resulting in very hydrophilic peptide. Such highly hydrophilic sequences have the strong antigenicity and are correspondingly likely to adopt a highly exposed conformation in the three-dimensional folding of a protein.
The Bovine Papillomavirus type 1 (BPV-1 E2) E2 protein is the master regulator of the viral life cycle—this protein modulates the transcription of viral genes (Spalholtz et al., Cell 42:183-191, 1985) and is responsible for the initiation of DNA replication (Ustav and Stenlund, EMBO J. 10:449-457, 1991) and for the stable maintenance of the viral genome (Piirsoo et al., EMBO J. 15:1-11, 1996; Ilves et al., J. Virol. 73:4404-4412, 1999). E2 is a sequence-specific DNA-binding protein, which is composed of three function-specific domains: the amino-terminal part (aa 1-200) is an activation domain for transcription and replication. It is followed by the unstructured hinge region and the carboxy-terminal DNA-binding and dimerization domain (aa 310-410) (Giri and Yaniv, EMBO J. 7:2823-2829, 1988). The protein is about 48 kDa and is 410 amino acid residues in length.